4 research outputs found
Contribution of Lsh to DNA methylation during neurodevelopment
DNA methylation is considered one of the oldest epigenetic modifications and is associated
with repression of transcriptional activity. Embryonic development sees dynamic changes in
this mark. Initially, widespread demethylation occurs with the establishment of pluripotency in
the developing zygote. This is then followed by a wave of de novo methylation in the
implanting blastocyst, coincident with lineage specification, which continues until tissue
specific patterns of DNA methylation are laid down.
Disruptions in DNA methylation, and mutations in genes that form part of the DNA
methylation machinery, are associated with neurodevelopmental disorders. Therefore,
understanding the mechanisms of DNA methylation during neurodevelopment is key if we
are to understand the pathology underlying these conditions. Investigating this has, thus far,
proven difficult due to the lack of available models that survive into adulthood. Therefore the
consequences of dysregulated DNA methylation during neurodevelopment on the mature
brain are unknown.
The DNA methyltransferase enzymes are responsible for the deposition of DNA methyl
marks, although less is known about the cofactors required for their targeting and activity.
Lsh (lymphoid specific helicase), a chromatin remodeller, has been described to play a key
role in de novo methylation during development. The importance of this protein is highlighted
by the fact that knockout mice die within a few hours of birth. Furthermore, whilst I was
pursuing this project, Thijsen et al described mutations in this gene being causative for
Immunodeficiency, Centromeric instability and Facial anomalies syndrome (ICF), in which a
large proportion of patients suffer from intellectual disability (Thijssen et al., 2015).
At the beginning of this project, nothing was known about the role of Lsh during
neurodevelopment, nor why mutations in this gene should result in neurological defects. This
thesis aims to determine the contribution of Lsh to DNA methylation during
neurodevelopment and to investigate the consequences of its absence on the mature brain.
In order to investigate the roles of Lsh at early stages of neurogenesis I made use of an in
vitro neurodifferentiation system to investigate the differentiation of Lsh-/- mouse embryonic
stem cells down neural lineage. This revealed an enhanced differentiation of these cells
down neural lineage compared to wild type. Genome-wide methylome analysis uncovered a
key role for Lsh in establishing appropriate DNA methylation at repetitive sequences during
this developmental window.
Previous papers, investigating selected loci, have suggested a role for Lsh in methylating
promoters of single copy genes thereby controlling their transcription. By investigating
transcription genome–wide, I noted a key role for Lsh in regulating genes associated with
developmental processes. This regulation was not, however, related to their promoter
methylation status. This led me to investigate the regulation of the Polycomb system in these
cells, another key repressive epigenetic system involved in developmental gene
transcriptional regulation.
This analysis uncovered wide-scale redistribution of the polycomb mark H3K27me3 from
target genes to hypomethylated repeats, similar to what is seen in other hypomethylated
models. Whilst this could account for the dysregulation of developmental gene expression, I
also discovered preliminary evidence of a role for Lsh in regulating transcription at these
sites independent of its role in DNA methylation.
To investigate the role of Lsh in vivo, I used a novel Nestin-Cre knockout mouse model. This
new targeted mouse model, unlike previous models, survived to adulthood. This allowed
investigation of the effects that disruption of DNA methylation during development has on the
mature brain. This mouse displayed no gross changes in brain morphology or behaviour.
DNA methylation analysis revealed gross hypomethylation at repeat sequences particularly
in the knockout cerebellum. A consequence of this was aberrant repeat transcription. RNASeq
revealed activation of innate immune response genes. This led me to propose a model
by which aberrant repeat transcription results in an immune response due to cellular
detection of cytoplasmic double stranded DNA generated via reverse transcription. This
opens up exciting new avenues of research into the underlying pathology of the neurological
deficits seen in ICF syndrome and potential therapeutic options